Histology analysis

For histological analysis, part of heart tissues were fixed in 4% paraformaldehyde (PFA) at 4°C overnight, then dehydrated and embedded in paraffin for preparation of 5-μm histological sections. Rehydrated slides were stained with haematoxylin and eosin (HE) staining to evaluate cardiomyocyte area and picric-sirius red staining to measure fibrosis area. Another part of heart tissues were fixed in 20% sucrose solution at 4°C overnight and embedded in O.C.T. compound for preparation of 8-μm frozen sections. Detection of senescent cells was determined in cells and frozen sections with the senescence β-galactosidase staining kit (GenMed Scientifics Inc.U.S.A) and lipofuscin staining kit (GenMed Scientifics Inc.U.S.A). Samples were analyzed by two independent investigators in a blind fashion.